Yeast TATA-box transcription factor gene.
Yeast TATA-box transcription factor gene.
复制标题
酵母 TATA-box 转录因子基因。
DOI：
10.1073/pnas.86.20.7785
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发表时间：
1989
影响因子：
11.1
通讯作者：
Berk,AJ
中科院分区：
文献类型：
--
作者：
Schmidt,MC;Kao,CC;Pei,R;Berk,AJ
真核生物中大多数编码蛋白质的基因转录的第一步是转录因子与TATA-box启动子元件的结合。该TATA-box转录因子是从酿酒酵母提取物中通过体外转录反应重组的方法纯化得到的。其活性与十二烷基硫酸钠/聚丙烯酰胺凝胶迁移率为25 kDa的蛋白质相互作用。该蛋白的氨基末端21个残基的序列由连续Edman降解法确定。用编码该蛋白质序列6个残基的混合寡核苷酸筛选酵母基因组文库。克隆了酵母Tata-box因子基因，DNA测序显示有一个720碱基的开放阅读框，编码一个27,016-Da的蛋白质。通过在大肠杆菌中表达该基因，并检测重组大肠杆菌提取物中TATA-box因子的DNA结合活性和转录活性，证实了该克隆的真实性。将TATA-box因子基因定位于酿酒酵母5号染色体上。RNA印迹杂交和核酸酶S1分析表明，TATA-box因子mRNA的主要片段为1.3kb，含有一个188+/-5个核苷酸的5‘非翻译区。同源搜索显示有一个区域与钙结合蛋白的钙结合结构有很远的相似之处，该结构的构象类似于DNA结合蛋白的螺旋-转角-螺旋基序。
The first step in the transcription of most protein-encoding genes in eukaryotes is the binding of a transcription factor to the TATA-box promoter element. This TATA-box transcription factor was purified from extracts of the yeast Saccharomyces cerevisiae by using reconstitution of in vitro transcription reactions as an assay. The activity copurified with a protein whose sodium dodecyl sulfate/polyacrylamide gel mobility is 25 kDa. The sequence of the amino-terminal 21 residues of this protein was determined by sequential Edman degradation. A yeast genomic library was screened with mixed oligonucleotides encoding six residues of the protein sequence. The yeast TATA-box factor gene was cloned, and DNA sequencing revealed a 720-base-pair open reading frame encoding a 27,016-Da protein. The identity of the clone was confirmed by expressing the gene in Escherichia coli and detecting TATA-box factor DNA binding and transcriptional activities in extracts of the recombinant E. coli. The TATA-box factor gene was mapped to chromosome five of S. cerevisiae. RNA blot hybridization and nuclease S1 analysis indicated that the major TATA-box factor mRNA is 1.3 kilobases, including an unusually long 5' untranslated region of 188 +/- 5 nucleotides. Homology searches showed a region of distant similarity to the calcium-binding structures of calpains, a structure that has a conformation similar to the helix-turn-helix motif of DNA binding proteins.